Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.     

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.     

Single electron reduction of 'slow' and 'fast' cytochrome-c oxidase.

Evidence is presented that single electron reduction is sufficient for rapid electron transfer (k greater than 20 s-1 at pH 8.0 in 0.43 M potassium EDTA) between haem a/CuA and the binuclear centre in 'fast' oxidase, whereas in 'slow' oxidase intramolecular electron transfer is slow even when both CuA and haem a are reduced (k congruent to 0.01 s-1). However, while a single electron can equilibrate rapidly between CuA, haem a and CuB in 'fast' oxidase, it seems that equilibration with haem a3 is relatively slow (k congruent to 2 s-1). Electron transfer between cytochrome c and CuA/haem a is similar for both types of enzyme (k congruent to 2.4 x 10(5) M-1.s-1).     

The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms

The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed.     

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family.

BACKGROUND: Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS: Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS: This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.     

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state.     

Electrochemical investigation of cellobiose oxidation by cellobiose dehydrogenase in the presence of cytochrome c as mediator

An important aspect of the cytochrome c electrochemistry is the possibility of coupling the 'heterogeneous reactions' with other redox enzymes. Cellobiose dehydrogenase, a 89170 Da glycoprotein that contains both FAD and a b-type haem as prosthetic groups, donates electrons to a number of acceptors, including cytochrome c. While haem b is surrounded mainly by acidic amino acids, cytochrome c displays positive charged lysine groups around the haem site. Thus a fast reaction between both proteins is explicable. In the presence of cellobiose, a catalytic current was observed, owing to the interaction of cellobiose dehydrogenase with electrostatically adsorbed cytochrome c. Adsorption of cytochrome c provides a technological model surface for vectorial electron transfer.     

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.     

Why do c-type cytochromes exist? Reprise.

An earlier paper (Wood, P.M. (1983) FEBS Lett. 164, 223-226) proposed that the covalent links that characterize c-type haem originated in order to prevent the haem being lost into the external medium. This is discussed in relation to Gram-negative and Gram-positive bacteria, cyanobacterial thylakoids and eukaryotes. The practical application is for unravelling complex electron transfer chains: c-type cytochromes may be assumed to be absent from the cytoplasm. Sulphate reducing bacteria provide the only confirmed exception to this rule. Examples of non-covalent haem at risk of being lost are considered. Analogies are drawn with flavin and pyrroloquinoline quinone as prosthetic groups.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the "prepumped" proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different Em values of the electron acceptor in the binuclear site.     

Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.

The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome.     

Comparison of the processes involved in reduction by the substrate for two homologous flavocytochromes b2 from different species of yeast.

A detailed study of the electron exchanges involved between FMN and haem b2 groups within flavocytochrome b2 of yeast Hansenula anomala (H-enzyme) was performed. The results were compared with those for the homologous enzyme of yeast Saccharomyces cerevisiae (Sx-enzyme) re-investigated at 5 degrees C. The mid-point reduction potentials of FMN and haem were determined by two complementary methods: potentiometric titration with substrate, L-lactate, in the presence of dye mediators with quantification of the reduced species performed by spectrophotometry at suitable wavelengths; anaerobic titration of the enzyme by its substrate by monitoring the e.p.r. signals of the semiquinone and Fe3+ species. Values of Em,7 = -19, -23 and -45 V were determined respectively from the data for the three redox systems Ho/Hr, Fo/Fsq and Fsq/Fr in the H-enzyme instead of +6, -44 and -57 mV respectively in the Sx-enzyme [Capeillère-Blandin, Bray, Iwatsubo & Labeyrie (1975) Eur. J. Biochem. 54, 549-566]. Parallel e.p.r rapid-freezing and absorbance stopped-flow studies allowed determination of the time courses of the various redox species during their reduction by L-lactate. The flavin and the haem reduction time courses were biphasic. In the initial fast phase the reduction of flavin monitored by absorbance measurements is accomplished with a rate constant kF = 360 s-1. The reduction of the haem lags the reduction of flavin with a rate constant kH = 170 s-1. The appearance of flavin free radical is slower than the reduction in flavin absorbance and occurs with a rate constant close to that of the reduction of the haem. At saturating L-lactate concentration the initial rapid phase (up to 15 ms) involved in the overall turnover can be adequately simulated with a two-step reaction scheme. The main difference between the enzymes lies especially at the level of the first step of electron exchange between bound lactate and flavin, which for the H-enzyme is no longer the rate-limiting step in the haem reduction and becomes 8-fold faster than in the Sx-enzyme. Consequently in the H-enzyme for the following step, the intramolecular transfer from flavin hydroquinone to oxidized haem, a reliable evaluation of the rate constants becomes possible. Preliminary values are k+2 = 380 s-1 and k-2 = 120 s-1 at 5 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Flavocytochrome b2: simulation studies of the electron-transfer reactions among the prosthetic groups.

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from L-lactate to flavocytochrome b2, which were presented in a preceding paper in this journal. The reaction scheme proposed as the "best" one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized flavin (Flox), the flavin semiquinone (Flsq), the fully reduced flavin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200-ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between flavin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein.     

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

 Reduction of the haems in tetrahaem cytochromes c ₃ is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c ₃ form a structural motif that is present in all cytochromes c ₃ and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c ₃. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c ₃. NMR studies of F20I cytochrome c ₃ demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c ₃ backbone, which is also very close to haem I, had no effect on the network of cooperativities. Received: 25 June 1996 / Accepted: 26 August 1996     

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

 The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H₂O₂ to H₂O with oxidation of cytochrome c ₅₅₀ and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4′-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca²⁺ is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem. Received: 29 April 1998 / Accepted: 3 September 1998     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system.

In anaerobic environments the first electron transfer in substrate-free P450cam is known to be thermodynamically unfavourable, but in the presence of dioxygen the reduction potential for the reaction shifts positively to make electron transfer thermodynamically favourable. Nevertheless a slower rate of electron transfer is observed in the substrate-free P450cam compared to substrate-bound P450cam. The ferric haem centre in substrate-free P450cam changes from six co-ordinate to five co-ordinate when reduced whereas in substrate-bound P450cam the iron centre remains five co-ordinate in both oxidation states. The slower rate of electron transfer in the substrate-free P450cam is therefore attributed to a larger reorganisation energy as predicted by Marcus theory.     

The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin.

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues. The protein is identical [as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f purified from turnip leaves. Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.